L-threonine dehydrogenase. Purification and properties of the homogeneous enzyme from Escherichia coli K-12.
L-Threonine dehydrogenase, which catalyzes the conversion of L-threonine to aminoacetone + CO2 presumably via the intermediate formation of alpha-amino-beta-ketobutyrate, has been purified to apparent homogeneity from extracts of a mutant of Escherichia coli K-12 which has constitutively derepressed levels of the enzyme. Three fractionation steps were used including controlled heat denaturation, DEAE-Sephadex chromatography, and blue dextran-Sepharose affinity chromatography. The purified enzyme migrated as a single band, coincident with dehydrogenase activity, when electrophoresed on polyacrylamide gels at pH 8.0 and 9.5. Electrophoresis in 1% sodium dodecyl sulfate also showed one band and a single schlieren peak was seen during sedimentation velocity centrifugation. The enzyme has an apparent molecular weight of 140,000 +/- 4,000 as determined by sucrose density and sedimentation equilibrium centrifugation. Based on electrophoresis in 1% sodium dodecyl sulfate, sedimentation equilibrium centrifugation in 6 M guanidine.HCl, and cross-linking with dimethyl suberimidate, the molecule is a tetramer consisting of identical (or nearly identical) subunits with Mr approximately equal to 35,000. L-Threonine dehydrogenase is specific for NAD+ or NAD+ analogs and utilizes L-threonine, D-allothreonine, or L-threonine amide as the best substrates. In 50 mM Tris.HCl buffer (pH 8.4) and 37 degrees C, the Km values for L-threonine and NAD+ are 1.43 and 0.19 mM, respectively. The enzyme has a pH optimum of 10.3, is activated by Mn2+, and shows a substantial loss of activity when treated with certain sulfhydryl-reacting reagents.
D000596:Amino Acids / Q000032:analysis; D004926:Escherichia coli / Q000201:enzymology*; D005779:Immunodiffusion; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D013379:Substrate Specificity; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013913:Threonine Dehydratase / Q000302:isolation & purification* / Q000378:metabolism
D000596:Amino Acids; D046911:Macromolecular Substances; D013439:Sulfhydryl Reagents; D013913:Threonine Dehydratase